The long-term objectives of my research are to characterize the properties of calcium release from the sarcoplasmic reticulum (SR) in skeletal muscle and determine the mechanism which links depolarization of the transverse tubular system to release of calcium from the SR during excitation. The specific aims of this project are to: i) demonstrate that inactivation of calcium release is calcium-dependent and determine if inactivation occurs in a restricted space at the release site; ii) develop and test a kinetic model for calcium-dependent inactivation; iii) determine the underlying molecular mechanism of inactivation; iv) study the effects of elevated (Ca2+) on charge movement and v) use this model of inactivation of SR calcium release in skeletal muscle to stimulate the properties of calcium release and uptake in other preparations. The rate of release of calcium will be calculated from (Ca2+) transients measured in single, cut skeletal muscle fibers from frog voltage clamped in a double Vaseline gap chamber. The indicator Antipyrylazo III will be used to measure changes in myoplasmic (Ca2+) from which the rate of calcium release will be calculated. The methodology will be developed to rapidly step the myoplasmic (Ca2+) using "caged calcium" and used in a variety of experiments to study the mechanism of inactivation. A preliminary model for calcium-dependent inactivation will be tested and the underlying mechanism of action explored by studying the effects of calmodulin and calmodulin inhibitors on the characteristics of inactivation. The inactivation model will be incorporated into a general model for calcium-induced calcium release. Simulations of calcium release and uptake in various preparations will be carried out and the effects of ruthenium red, (Mg2+), nucleotides, and ryanodine on release will be simulated. A model for calcium oscillations in cardiac cells will be developed and tested.